1. Field of Invention
The present invention pertains to peptide substrates of a proteolytic ADAM33 polypeptide. In addition, the present invention pertains to identification of additional substrates of a proteolytic ADAM33 polypeptide. Furthermore, the present invention pertains to the use of these peptide substrates for identifying compounds that inhibit the proteolytic activity of a proteolytic ADAM33 polypeptide.
2. Background
Asthma is a chronic respiratory disorder that afflicts hundreds of millions of people throughout the world [Drazen and Weiss, Nature, 418(6896):383–384 (2002)]. Though the occurrence of this respiratory disorder has been noted for over two thousand years, during the past twenty years industrialized nations have experienced an increase in asthma sufferers that approaches epidemic proportions [Umetsu et al., Nat Immunol, 3(8):715–720 (2002)]. Indeed, 10–20% of the population of industrialized countries currently suffer from asthma. Not surprisingly, the dramatic increase in the number of asthmatics in industrialized nations has resulted in a concomitant expenditure of resources to treat this condition [Umetsu et al., Nat Immunol, 3(8):715–720 (2002)]. Despite this strong commitment, to date the treatments employed only control the symptoms.
Asthma is characterized by life-threatening attacks due to episodic obstructions to, or abnormal narrowing of the airways in response to otherwise innocuous stimuli [Drazen and Weiss, Nature, 418(6896):383–384 (2002)]. Common symptoms of asthma include recurrent episodes of coughing, wheezing and breathlessness. The immediate cause for the thickening of the airway walls, smooth muscle contraction, and narrowing of the airways observed in asthmatics is an inflammation mediated by T-cells [Van Eerdewegh et al., Nature, 418(6896):426–430 (2002)]. Both genetic and environmental factors play key roles in inducing this T-cell-mediated inflammation, though the actual mechanism has yet to be delineated. What is known is that asthmatics have a genetic predisposition for the disease, and environmental factors serve to either trigger or protect against this immunological dysregulation [Umetsu et al., Nat Immunol, 3(8):715–720 (2002)].
Recently, the gene encoding a membrane anchored protein known as ADAM33 has been shown to be linked to asthma by positional cloning in an outbred population [Van Eerdewegh et al., Nature, 418(6896):426–430 (2002)]. Both human and mouse ADAM33 have been identified and characterized [Yoshinaka et al., Gene, 282(1–2):227–236 (2002); Gunn et al., BMC Genet, 3(1):2, (2002)].
ADAM33, expressed primarily in human lung fibroblasts and bronchial smooth muscle [Van Eerdewegh et al., Nature, 418(6896):426–430 (2002)], is a member of the “A Disintegrin And Metalloprotease” (ADAM) family of proteins. The ADAM family of proteins comprises over thirty such proteins, including the well characterized TNF-alpha converting enzyme (TACE) [Cross et al., J Am Chem Soc, 124(37):11004–11007 (2002); Schlondorff and Blobel, J Cell Sci, 112(Pt 21):3603–3617 (1999); Black, Int J Biochem Cell Biol, 34(1):1–5 (2002); U.S. Pat. No. 5,830,742]. The ADAM family of proteins is a class of type-I transmembrane proteins that share a unique domain structure composed of a signal sequence, a prodomain, a metalloprotease/catalytic domain, a disintegrin domain, a cysteine-rich domain, an epidermal growth factor-like domain, a transmembrane and a cytoplasmic domain. Though, both human and mouse ADAM33 have been identified and sequenced, heretofore, little specific information has been provided regarding their catalytic activity. Moreover, the ADAM33 protein domains, including the catalytic domain, have not been specifically delineated and isolated.
Due to its genetic linkage to asthma and expression pattern, ADAM33 has become a promising target protein for use in identifying compounds to treat asthma [Shapiro and Owen, N Engl J Med, 347(12):936–938 (2002)]. Prior to this invention, no substrate of a proteolytic ADAM33 polypeptide had been identified. In particular, the identification of substrates of a proteolytic ADAM33 polypeptide is important for assaying its proteolytic activity. In addition, substrates of a proteolytic ADAM33 polypeptide would be useful for assaying compounds that modulate the proteolytic activity of a proteolytic ADAM33 polypeptide. In particular, compounds that inhibit the proteolytic activity of a proteolytic ADAM33 polypeptide may be useful in the treatment of asthma.
Therefore, there is a need for substrates of a proteolytic ADAM33 polypeptide. In addition, there is a need to provide methods for identifying compounds that are substrates of a proteolytic ADAM33 polypeptide. Furthermore, there is a need to provide methods for identifying compounds that inhibit the proteolytic activity of a proteolytic ADAM33 polypeptide.